In human cells, the enzymes thymidin phosphorylase
(TP), dihydropyrimidine dehydrogenase (DPD), and thymidylate
synthase (TS) play a pivotal role in the metabolism
of pyrimidines. In cancer disease medical care, the
cytotoxic compound 5-fluorouracil (5-FU) is the standard
chemotherapy treatment for a wide range of solid
tumors. Attempts to increase the efficacy and tolerability
of fluoropyrimidine treatment has led to the development
of Capecitabine (Xeloda), a fluoropyrimidine drug
from Roche Pharmaceuticals. Capecitabine is not cytotoxic
unless it is activated to 5-FU by TP a three-step
mechanism. Therefore, it has several advantages over
ordinary 5-FU by treatment [1, 2].

Capecitabine is preferentially activated and converted to
5-FU due to TP upregulation at the tumor site. 5-FU
inhibits TS, an enzyme crucial for the de novo synthesis
of thymidine nucleotides. The rate-limiting enzyme in the
catabolism of 5-FU is DPD, which catalyzes the first step
of pyrimidine degradation (Figure 1).

Efficacy and tolerability of Capecitabine or similar drugs
may therefore largely depend on the expression rates of
each of the three enzymes. These expression rates can
be estimated by the detection of the relative mRNA
expression levels of the three enzymes in tumor tissue or
blood [3], using the new LightCycler TP, DPD, and TS
mRNA Quantification KitsPLUS.

Materials and Methods

Fresh-frozen tissue and paraffin-embedded tissue (PET)
slides (5 m) were taken from human xenografts in
mouse models. Isolation of RNA was performed using
the MagNA Lyser Instrument and the High Pure RNA
Paraffin Kit. The human RNA panel was obtained from
Clontech.
Based on the LightCycler mRNA Quantification Kit
concept, we have developed three new reagent kits for
TP, DPD, and TS that are specially designed for usage
with fresh-frozen tissues or PET in research applications.
The kits are based on a two-step protocol for the quantification of target mRNA expression rates relative
to a reference gene (glucose-6-phosphate dehydrogenase,
G6PDH) using the LightCycler Instrument and the
Relative Quantification Software.
In the first step (reverse transcription), gene-specific
primers are utilized to obtain TP, DPD, TS, and G6PDH
cDNA in separate reactions. Gene-specific priming is
necessary as RNA from PET is degraded to fragments
smaller than approximately 200 bp.
The cDNA is then used as a template for quantitative
PCR using the LightCycler Hybridization Probe format.
Additionally, calibrator RNA from an immortalized cell
line is included in the kit and is used as an external standard
to allow for relative quantification in each run.

Results and Applications

To validate the new kit concept we have performed three
comparative studies:

Proof of principle specific priming

With the standard primer concentration (1 M), the amplification
curves in cDNA synthesis tend to decline with decreasing
template concentration (Figure 2). Reducing the
concentration of the specific primers to 1/5 (0.2 M) leads
to reproducible and stable curves in the reverse transcription-
polymerase chain reaction (RT-PCR) dilution series,
especially with low RNA concentrations (Figure 2). Template
RNA in the range of 500 ng/l to as little as 32 pg/l
was used. TP data are shown as being representative for
DPD, TS, and G6PDH using calibrator RNA as a template.

The new primer concentration also extends the dynamic
measuring range of the target and reference and improves
sensitivity compared to random hexamer priming (Figure 3
a-c). Table 3d numerically shows the crossing point (Cp)
ranges for all four genes TP, DPD, TS, and G6PDH.

The human RNA panel (healthy tissue)

We have also analyzed both priming concepts using a
panel of 21 human RNA samples from healthy tissues. We observed a slight shift to smaller Cp values with
specific priming for all four parameters TP, DPD, TS
and G6PDH indicating the higher sensitivity of the new
concept (Figure 4). These shifts depend on the parameter
and the analyzed tissue type. Also, the comparison
of the relative ratios reveals a tendency for slightly
higher ratios with specific priming. This is caused by
different Cp shifts between calibrator and sample
RNA. Nevertheless, the expression rates are comparable
and within a common range.

Comparison of fresh-frozen tissue vs. PET

For this study, we analyzed xenografts from human
immortalized colon tumor cell lines HT29 and SW62
with the new kit concept. Total RNA was isolated from
PET in 4 x 5-m slides and from matched fresh-frozen
tissues in two 20-mg slices according to the High Pure
RNA Paraffin Kit protocol. As depicted in Figure 5, the
relative expression rates of TP, DPD, and TS are comparable
in both tissue types.

The new kit concept with specific priming is superior to
former hexamer protocols and shows comparable Cp
values and ratios. The linear measuring ranges were
extended and the sensitivity of analysis was improved
for all three target genes, as well as for the reference
gene G6PDH. The analysis of matched fresh-frozen tissues
and PET yields nearly identical relative expression
rates.

These preliminary and preclinical results suggest that
the LightCycler Quantification KitsPLUS for TP, DPD and
TS are well suited for the analysis of fresh-frozen tissue
samples, as well as for retrospective investigations with
PET samples in research applications.

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